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Non-tuberculous mycobacteria (NTM) are considered a relevant cause of non-specific reactions to the most widely applied bovine tuberculosis (bTB) test, the intradermal tuberculin test. In order to establish which NTM species might act as a potential source of such diagnostic interference, a collection of 373 isolates obtained from skin test positive cows from 359 officially tuberculosis-free (OTF) herds, culled in the framework of the bTB eradication campaign in Spain, were identified at the species level through PCR and Sanger sequencing of the 16S rDNA, hsp65 and rpoB genes.Of the 308 isolates for which a reliable identification was achieved, 32 different mycobacterial species were identified, with certain species being most represented: among M. avium complex members (n = 142, 46.1%), M. aviumsubsp. hominissuis (98; 69.0%) was themost abundant followed by M. avium subsp. avium (33, 23.2%), and M. intracellulare (7, 4.9%). Among non-MAC members (n = 166, 53.9%), M. nonchromogenicum(85; 27.6%) and M. bourgelatii (11; 5.6%) were the predominant species. In addition, mixed results were obtained in 53 isolates presenting up to 30 different genotypes, which could be indicative of new mycobacterial species. Our results represent a first step toward characterizing the diversity of NTM species that could interfere with official diagnostic tests for bTB eradication in Spain. [ABSTRACT FROM AUTHOR]

The diagnostic methods for granting and maintenance of the official tuberculosis-free (OTF) status and for intra-Community movement of cattle are the tuberculin skin tests (single or comparative) and the interferon-γ (IFN-γ) release assay (IGRA). However, until now, IGRAs have been primarily applied in infected farms in parallel to the skin test to maximize the number of infected animals detected. Therefore, an evaluation of the performance of IGRAs in OTF herds to assess whether if their specificity is equal to or higher than that of the skin tests is needed. For this, a panel of 4365 plasma samples coming from 84 OTF herds in six European regions (five countries) was assembled and analysed using two IGRA kits, the ID Screen® Ruminant IFN-g (IDvet) and the Bovigam™ TB Kit (Bovigam). Results were evaluated using different cut-offs, and the impact of herd and animal-level factors on the probability of positivity was assessed using hierarchical Bayesian multivariable logistic regression models. The percentage of reactors ranged from 1.7 to 21.0% (IDvet: S/P ≥ 35%), and 2.1–26.3% (Bovigam: ODbovis–ODPBS ≥ 0.1 and ODbovis–ODavium ≥ 0.1) depending on the region, with Bovigam disclosing more reactors in all regions. The results suggest that specificity of IGRAs can be influenced by the production type, age and region of origin of the animals. Changes in the cut-offs could lead to specificity values above 98–99% in certain OTF populations, but no single cut-off yielding a sufficiently high specificity (equal or higher than that of skin tests) in all populations was identified. Therefore, an exploratory analysis of the baseline IFN-γ reactivity in OTF populations could help to assess the usefulness of this technique when applied for the purpose of maintaining OTF status. [ABSTRACT FROM AUTHOR]

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity. [ABSTRACT FROM AUTHOR]

Despite the efforts invested in the eradication of bovine tuberculosis in Spain, herd prevalence has remained constant in the country during the last 15 years (~1.5-1.9%) due to a combination of epidemiological factors impairing disease control, including between-species transmission. Here, our aim was to investigate the molecular diversity of Mycobacterium bovis isolates belonging to the highly prevalent SB0339 spoligotype in the cattle-wildlife interface in different regions of Spain using whole-genome sequencing (WGS). Genomic data of 136 M. bovis isolates recovered from different animal species (cattle, wild boar, fallow deer, and red deer) and locations between 2005 and 2018 were analyzed to investigate between- and withinspecies transmission, as well as within-herds. All sequenced isolates differed by 49-88 single nucleotide polymorphisms from their most recent common ancestor. Genetic heterogeneity was geographic rather than host species-specific, as isolates recovered from both cattle and wildlife from a given region were more closely related compared to isolates from the same species but geographically distant. In fact, a strong association between the geographic and the genetic distances separating pairs of M. bovis isolates was found, with a significantly stronger effect when cattle isolates were compared with wildlife or cattle-wildlife isolates in Spain. The same results were obtained in Madrid, the region with the largest number of sequenced isolates, but no differences depending on the host were observed. Within-herd genetic diversity was limited despite the considerable time elapsed between isolations. The detection of closely related strains in different hosts demonstrates the complex between-host transmission dynamics present in endemic areas in Spain. In conclusion, WGS results a valuable tool to track bTB infection at a high resolution and may contribute to achieve its eradication in Spain. [ABSTRACT FROM AUTHOR]

Even though enormous efforts and control strategies have been implemented, bovine tuberculosis (TB) remains a significant source of health and socioeconomic concern. The standard method used in TB eradication programs for in vivo detection is the tuberculin skin test. However, the specificity of the tuberculin skin test is affected by infection with non‐tuberculous mycobacteria or by vaccination. Thus, some animals are not correctly diagnosed. This study aimed first to identify a plasma metabolic TB profile by high‐field (HF) nuclear magnetic resonance (NMR) spectroscopy and second measure this characteristic TB metabolic profile using low‐field benchtop (LF) NMR as an affordable molecular technology for TB diagnosis. Plasma samples from cattle diagnosed with TB (derivation set, n = 11), diagnosed with paratuberculosis (PTB, n = 10), PTB‐vaccinated healthy control (n = 10) and healthy PTB‐unvaccinated control (n = 10) were analyzed by NMR. Unsupervised Principal Component Analysis (PCA) was used to identify metabolic differences between groups. We identified 14 metabolites significantly different between TB and control animals. The second group of TB animals was used to validate the results (validation set, n = 14). Predictive models based on metabolic fingerprint acquired by both HF and LF NMR spectroscopy successfully identified TB versus control subjects (Area under the curve of Receiver Operating Characteristic over 0.92, in both models; Confidence Interval 0.77–1). In summary, plasma fingerprinting using HF and LF‐NMR differentiated TB subjects from uninfected animals, and PTB and PTB‐vaccinated subjects who may provide a TB‐false positive, highlighting the use of LF‐NMR‐based metabolomics as a complementary or alternative diagnostic tool to the current diagnostic methods. [ABSTRACT FROM AUTHOR]

Between‐farm transmission of bovine tuberculosis (bTB) occurs mostly through fence‐to‐fence contact between neighbouring farms, endemic infected wildlife or movement of infected animals. Unfortunately, bTB detection is frequently delayed and identification of the source of introduction is often difficult, particularly in endemic regions. Here, we characterized the cattle movement network of Castilla y Leon, a high bTB‐prevalence (1.9% at the farm level in 2015) region in Spain, over six years and analysed the distribution of bTB to ultimately assess the likelihood of spatial and movement‐mediated transmission. We analysed movement and bTB data from 27,633 units located in the region, of which 87% were involved in ~1.4 million movements of ~8.8 million animals. Network‐level connectivity was low, although a few highly connected units were identified. Up to 15% of the herds became bTB‐positive at some point during the study, with the highest percentage found in bullfighting and beef herds. Although bTB‐positive herds had a significantly higher degree and moved more cattle than negative herds. Results of the k‐test, a permutation‐based procedure, suggested that positive farms were not significantly clustered in the movement network. Location was a likely risk factor as bTB‐positive farms tended to be located within 5 km from each other. Results suggested that movements may be a source of bTB in cattle in Castilla y Leon, although local factors may be more influential in determining risk of disease at the farm level. The description of the movement network in Castilla y Leon may be valuable for bTB surveillance in Spain. Moreover, results are useful to assess the movement‐associated risk for multiple diseases. [ABSTRACT FROM AUTHOR]

Background: The genetic diversity of M. bovis in Tunisia is still underestimated despite the implementation of an eradication program. The lack of data about spatial distribution of the M. bovis population hinders the control of bovine tuberculosis (bTB) progress. This study represents the largest molecular analysis of M. bovis isolates in Tunisia. It is aimed to upgrade the understanding of bTB epidemiology and the geographical distribution of the infection. Tuberculosis research was performed in cattle (n = 149) with TB-compatible lesions collected over 5 months from a slaughterhouse located in Sfax, Tunisia. Results: Ninety-four animals were found to be infected by M. bovis and two others by M. caprae. Spoligotyping revealed twenty-five patterns, SB0120, SB0134, and SB0121 being the most prevalent profiles (36.4%, 11.4%, and 7.2%, respectively). Three new spoligotypes were detected: SB2345, SB2344 and SB2343. MIRU-VNTR analysis classified the isolates in seventy-three profiles and showed a large genotypic variety observed within the main spoligotype which was split into several MIRU-VNTR types: 29 in SB0120 (h = 0.983), 10 in SB0134 (h = 0.981) and 7 in SB0121 (h = 1). Genotyping revealed a common pattern in different geographic regions. It also showed that Sfax, located in southern-Tunisia, represents a high-risk area with an elevated genetic diversity. Conclusions: Spatial analysis may provide insights into disease transmission, which affects the effectiveness of eradication campaigns in cattle. [ABSTRACT FROM AUTHOR]

Background: Bovine purified protein derivative (bPPD) and avian purified protein derivative (aPPD) are widely used for bovine tuberculosis diagnosis. However, little is known about their qualitative and quantitative characteristics, which makes their standardisation difficult. In addition, bPPD can give false-positive tuberculosis results because of sequence homology between Mycobacterium bovis (M. bovis) and M. avium proteins. Thus, the objective of this study was to carry out a proteomic characterisation of bPPD, aPPD and an immunopurified subcomplex from bPPD called P22 in order to identify proteins contributing to cross-reactivity among these three products in tuberculosis diagnosis. Methods: Trypsin digests of bPPD, aPPD and P22 were analysed by nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry. Mice were immunised with bPPD or aPPD, and their serum was tested by indirect ELISA for reactivity against these preparations as well as against P22. Results: A total of 456 proteins were identified in bPPD, 1019 in aPPD and 118 in P22; 146 of these proteins were shared by bPPD and aPPD, and 43 were present in all three preparations. Candidate proteins that may cause crossreactivity between bPPD and aPPD were identified based on protein abundance and antigenic propensity. Serum reactivity experiments indicated that P22 may provide greater specificity than bPPD with similar sensitivity for ELISAtype detection of antibodies against M. tuberculosis complex. Conclusion: The subpreparation from bPPD called P22 may be an alternative to bPPD for serodiagnosis of bovine tuberculosis, since it shares fewer proteins with aPPD than bPPD does, reducing risk of cross-reactivity with anti-M. avium antibodies. [ABSTRACT FROM AUTHOR]

Progress in control of bovine tuberculosis (bTB) is often not uniform, usually due to the effect of one or more sometimes unknown epidemiological factors impairing the success of eradication programs. Use of spatial analysis can help to identify clusters of persistence of disease, leading to the identification of these factors thus allowing the implementation of targeted control measures, and may provide some insights of disease transmission, particularly when combined with molecular typing techniques. Here, the spatial dynamics of bTB in a high prevalence region of Spain were assessed during a three year period (2010–2012) using data from the eradication campaigns to detect clusters of positive bTB herds and of those infected with certain Mycobacterium bovis strains (characterized using spoligotyping and VNTR typing). In addition, the within-herd transmission coefficient (β) was estimated in infected herds and its spatial distribution and association with other potential outbreak and herd variables was evaluated. Significant clustering of positive herds was identified in the three years of the study in the same location (“high risk area”). Three spoligotypes (SB0339, SB0121 and SB1142) accounted for >70% of the outbreaks detected in the three years. VNTR subtyping revealed the presence of few but highly prevalent strains within the high risk area, suggesting maintained transmission in the area. The spatial autocorrelation found in the distribution of the estimated within-herd transmission coefficients in herds located within distances <14 km and the results of the spatial regression analysis, support the hypothesis of shared local factors affecting disease transmission in farms located at a close proximity. [ABSTRACT FROM AUTHOR]

Use of mathematical models to study the transmission dynamics of infectious diseases is becoming increasingly common in veterinary sciences. However, modeling chronic infectious diseases such as bovine tuberculosis (bTB) is particularly challenging due to the substantial uncertainty associated with the epidemiology of the disease. Here, the methodological approaches used to model bTB and published in the peer-reviewed literature in the last decades were reviewed with a focus on the impact that the models' assumptions may have had on their results, such as the assumption of density vs. frequency-dependent transmission, the existence of non-infectious and non-detectable stages, and the effect of extrinsic sources of infection (usually associated with wildlife reservoirs). Although all studies suggested a relatively low rate of within-herd transmission of bTB when test-and-cull programs are in place, differences in the estimated length of the infection stages, sensitivity and specificity of the tests used and probable type of transmission (density or frequency dependent) were observed. Additional improvements, such as exploring the usefulness of contact-networks instead of assuming homogeneous mixing of animals, may help to build better models that can help to design, evaluate and monitor control and eradication strategies against bTB. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (TB), mainly caused by Mycobacterium bovis, is a zoonotic disease with implications for Public Health and having an economic impact due to decreased production and limitations to the trade. Bovine TB is subjected to official eradication campaigns mainly based on a test and slaughter policy using diagnostic assays based on the cell-mediated immune response as the intradermal tuberculin test and the gamma-interferon (IFN-#947;) assay. Moreover, several diagnostic assays based on the detection of specific antibodies (Abs) have been developed in the last fewyearswith the aim of complementing the current diagnostic techniques in the near future. This review provides an overview of the current ante-mortem diagnostic tools for diagnosis of bovine TB regarding historical background, methodologies and sensitivity (Se) and specificity (Sp) obtained in previous studies under different epidemiological situations. [ABSTRACT FROM AUTHOR]

Background Despite great effort and investment incurred over decades to control bovine tuberculosis (bTB), it is still one of the most important zoonotic diseases in many areas of the world. Testand- slaughter strategies, the basis of most bTB eradication programs carried out worldwide, have demonstrated its usefulness in the control of the disease. However, in certain countries, eradication has not been achieved due in part to limitations of currently available diagnostic tests. In this study, results of in-vivo and post-mortem diagnostic tests performed on 3,614 animals from 152 bTB-infected cattle herds (beef, dairy, and bullfighting) detected in 2007-2010 in the region of Castilla y León, Spain, were analyzed to identify factors associated with positive bacteriological results in cattle that were non-reactors to the single intradermal tuberculin test, to the interferon-gamma (IFN-γ) assay, or to both tests applied in parallel (Test negative/Culture + animals, T-/C+). The association of individual factors (age, productive type, and number of herd-tests performed since the disclosure of the outbreak) with the bacteriology outcome (positive/negative) was analyzed using a mixed multivariate logistic regression model. Results The proportion of non-reactors with a positive post-mortem result ranged from 24.3% in the case of the SIT test to 12.9% (IFN-γ with 0.05 threshold) and 11.9% (95% CI 9.9-11.4%) using both tests in parallel. Older (>4.5 years) and bullfighting cattle were associated with increased odds of confirmed bTB infection by bacteriology, whereas dairy cattle showed a significantly lower risk. Ancillary use of IFN-γ assay reduced the proportion of T-/C + animals in high risk groups. Conclusions These results demonstrate the likelihood of positive bacteriological results in non-reactor cattle is influenced by individual epidemiological factors of tested animals. Increased surveillance on non-reactors with an increased probability of being false negative could be helpful to avoid bTB persistence, particularly in chronically infected herds. These findings may aid in the development of effective strategies for eradication of bTB in Spain. [ABSTRACT FROM AUTHOR]

Background: Eradication of bovine tuberculosis (bTB) through the application of test-and-cull programs is a declared goal of developed countries in which the disease is still endemic. Here, longitudinal data from more than 1,700 cattle herds tested during a 12 year-period in the eradication program in the region of Madrid, Spain, were analyzed to quantify the within-herd transmission coefficient (β) depending on the herd-type (beef/dairy/ bullfighting). In addition, the probability to recover the officially bTB free (OTF) status in infected herds depending on the type of herd and the diagnostic strategy implemented was assessed using Cox proportional hazard models. Results: Overall, dairy herds showed higher β (median 4.7) than beef or bullfighting herds (2.3 and 2.2 respectively). Introduction of interferon-gamma (IFN-γ) as an ancillary test produced an apparent increase in the β coefficient regardless of production type, likely due to an increase in diagnostic sensitivity. Time to recover OTF status was also significantly lower in dairy herds, and length of bTB episodes was significantly reduced when the IFN-γ was implemented to manage the outbreak. Conclusions: Our results suggest that bTB spreads more rapidly in dairy herds compared to other herd types, a likely cause being management and demographic-related factors. However, outbreaks in dairy herds can be controlled more rapidly than in typically extensive herd types. Finally, IFN-γ proved its usefulness to rapidly eradicate bTB at a herd-level. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium bovis is the main causative agent of bovine tuberculosis. This zoonotic disease produces important economic losses and must be considered a threat to endangered animal species and public health. This study was performed (1) to assess the degree of diversity of the Spanish M. bovis isolates and its effect on the epidemiology of the infection, and (2) to understand the connection of M. bovis populations within a European context. In this report we resume the DVR-spoligotyping results of 6215 M. bovis isolates collected between 1992 and 2007 from different hosts. The isolates clustered into 252 spoligotypes which varied largely in frequency, geographical distribution and appearance in different animal species. In general, the most frequent spoligotypes were found all over the country and in different animal species, though some were restricted to a geographical area. Among our most often isolated spoligotypes, SB0121 and SB0120 (BCG-like) are a common feature between mainland European countries, however, the spoligotypes differ with those found in the UK, the Republic of Ireland and abroad. A comparison of spoligotypes reported from other countries reveals hints for the M. bovis demography in Europe and suggests a common ancestor strain. This study gives insight into the usefulness of the standardized DVR-spoligotyping technique for epidemiological studies in a country with a high degree of strain diversity. [Copyright &y& Elsevier]

• Efficiency of bTB abattoir surveillance should be regularly monitored. • No information on performance of bTB abattoir surveillance in high prevalence areas of Spain is available. • Detection of lesions was highly dependent on animal and farm characteristics and season. • Large differences in the abattoir specific lesion detection rates were found. • Information here will be useful to establish baseline detection targets. Post-mortem inspection (PMI) of routinely slaughtered cattle in abattoirs is an extremely valuable tool for detecting bovine tuberculosis (bTB) infected herds that can supplement active surveillance activities. However, its true performance is difficult to assess due to the multiple factors that may affect it. Here, we determined relative efficiencies in the detection of bTB-compatible lesions and probabilities of subsequent laboratory confirmation of abattoirs located in Castilla y Leon, one of the regions with the largest cattle population in Spain, between 2010 and 2017. The slaughtered animal population was split based on the results of the ante-mortem tests (reactors or non-reactors), and two generalized linear multivariable mixed models were fitted to each subpopulation to calculate the risk of lesion detection and laboratory confirmation per abattoir while accounting for the effect of potential confounding variables. Throughout the 8-year period, ∼30,000 reactors and >2.8 million non-reactor animals in the ante-mortem tests were culled in the abattoirs under study. Bovine TB compatible lesions were detected in 4,710 (16%) reactors and 828 (0.03%) non-reactor animals, of which >95% were confirmed as infected through bacteriology. The probability of disclosure of bTB-like lesions was associated with the animal subpopulation, type of source unit, the herd size, the year of slaughter, the breed and age of the animal, and/or the season of slaughter. The probabilities of detection of bTB-like lesions varied largely depending on the abattoir in both subpopulations, ranging from 603 to 3,070 per 10,000 animals for the reactors and 0.2–16.1 per 10,000 animals for the non-reactor animals. Results obtained here will help to quantify the performance of PMI in abattoirs in Castilla y Leon and the between-abattoir variability, and to identify animals at increased risk of having bTB-like lesions detected during PMI based on animal- and farm-related factors. [ABSTRACT FROM AUTHOR]

Abstract: Spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis are the international standard techniques for molecular typing of members of the Mycobacterium tuberculosis complex. To enable the exploitation of molecular typing data for epidemiological purposes, the creation of large databases is indispensable. Here we describe mycoDB.es, a database for animal tuberculosis which forms part of the Spanish national programme for eradication of bovine tuberculosis. This database has been created as an epidemiological tool at national level and contains spoligotype patterns of 17,273 isolates clustered in 401 different spoligotypes of Mycobacterium bovis, Mycobacterium caprae and M. tuberculosis. The database offers an overview of the present spoligotypes, to a lower extent also of MIRU-VNTR types, affected animal species and furthermore of the spatial distribution of these genotypes. [Copyright &y& Elsevier]

• The herd-level sensitivity of the current bTB diagnostic strategy in Spain was assessed. • SIT test can detect 76.2% of newly bTB-infected herds in a first herd-test. • In 65.3% of newly infected-SIT positive herds the presence of MTC is confirmed by culture. • 8.7% of infected but not-confirmed herds may regain OTF status after two herd-tests. • Herd sensitivity of both SIT test and culture directly correlated with herd size. In spite of the efforts invested to eradicate bovine tuberculosis (bTB) in cattle, the disease is still present in several developed countries, including Spain. Among the factors related with disease persistence in high prevalence areas, the lack of sensitivity of the screening test for detection of infected herds [single intradermal tuberculin (SIT) test] can play a major role. Here, a scenario tree model mimicking the diagnostic test scheme in place in the region of Castilla y Leon (Spain) was developed to estimate the probability of detecting bTB in an infected-non detected officially tuberculosis free (OTF) herd (herd sensitivity, HSe). In order to do so the probability of detecting at least one positive animal in the SIT test with/without post-mortem (detection of lesions and culture) confirmation in an infected herd was estimated using Monte Carlo simulation through @RISK (Palisade Co, NY, USA). Uncertainty on the accuracy of the diagnostic tests was introduced in the model using distributions based on the literature. The performance of the model was evaluated by comparing the predicted number of SIT/post-mortem positive animals in infected herds with those observed in newly detected bTB-infected herds in the region in 2011–2015. The estimated HSe of the SIT test was 76.2% (95% probability interval: 19.8–97.6). According to the model, bTB infection would be then confirmed through culture in 65.3% (95% PI: 50.0–82.3) of the herds detected through the SIT test, so that overall the proportion of infected-non detected OTF herds in which the infection could be confirmed after the initial SIT test was 49.6% (95% PI: 9.75–80.3). The predicted HSe of both SIT test and culture was directly correlated with herd size. Results from the model suggest a moderate but highly variable HSe of the current surveillance system in place for bTB detection in OTF herds located in high prevalence areas, that could be maximized by performing multiple tests within a year as indicated in the Spanish eradication program (with a median SIT HSe of 87% when two consecutive tests were considered). In addition, these results highlight the usefulness of performing subsequent SIT tests to rule out infection in SIT-positive herds even when the causative agent cannot be isolated. [ABSTRACT FROM AUTHOR]